Method of inhibiting harmful microorganisms and barrier-forming composition therefor

ABSTRACT

A method for blocking, neutralizing, or killing microorganisms that cause infectious disease in a mammal prior to or during the mammal encountering a contaminated environment or item includes: identifying a contaminated environment or item, wherein the contaminated environment or item is known or expected to be contaminated with harmful viral, fungal, or bacterial microorganisms; and administering a therapeutically effective amount of a barrier-forming composition to a mucosa of the mammal prior to or during the mammal encountering the contaminated environment or item. The barrier-forming composition provides a barrier on the mucosa that inhibits the microorganisms from contacting the mucosa. Other related methods are also included.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/014,448, filed on Aug. 30, 2013, titled “Method of Inhibiting HarmfulMicroorganisms and Barrier-Forming Composition Therefor,” which, inturn, was a continuation of U.S. application Ser. No. 13/448,926, filedon Apr. 17, 2012, titled “Method of Inhibiting Harmful Microorganismsand Barrier-Forming Composition Therefor,” which, in turn, claimed thebenefit of priority to U.S. provisional application No. 61/477,147,filed on Apr. 19, 2011, entitled “Compositions, Methods of Use, andMethods of Making Barrier Products.” Each of these prior applicationsare incorporated herein by reference for all purposes.

FIELD

This disclosure relates to barrier-forming compositions and methods forpreventing communicable diseases.

BACKGROUND

There has been a longstanding need for devices, compositions, and othertreatments that will effectively prevent communicable diseases. Attemptsat solving this problem include wearing masks or respirators andavoiding or quarantining of individuals or animals that are known orexpected to be sick or carrying germs. Such approaches are common incertain countries where masks are worn by persons encounteringcontaminated environments such as public transportation or publicgathering places.

Other attempts to prevent infection have included large amounts of zinc,vitamins, or herbs that are theorized to work internally to boost thebody's immune system.

While numerous solutions exist for killing microorganisms once they havecontacted a person or animal, the effectiveness of such solutions isdependent on quick recognition of the germ contact and application ofthe germ-killing composition prior to the microorganism binding to amucosa, whereby it would enter the body and infect the individual. Forexample, washing with an anti-bacterial soap may be effective forkilling germs on the hands; however, it is very easy for a person tounwittingly touch a contaminated surface and put their hands near or intheir mouth or nose before washing their hands.

Physical devices such as masks are uncomfortable, zinc, vitamin C, andherbal remedies have unproven results, and solutions for killing germsthat have already contacted the body are often ineffective forprevention of infection since they are intermittent, transitory optionsthat do not provide sustained protection.

Compositions have been developed for forming blocking barriers topicallyon human skin or in the oral or internal cavities. However, suchcompositions are not for preventing infection of communicable diseases.

SUMMARY

In an embodiment, a method for blocking, neutralizing, or killingmicroorganisms that cause infectious disease in a mammal prior to orduring the mammal encountering a contaminated environment or itemincludes: identifying a contaminated environment or item, wherein thecontaminated environment or item is known or expected to be contaminatedwith harmful viral, fungal, or bacterial microorganisms; andadministering a therapeutically effective amount of a barrier-formingcomposition to a mucosa of the mammal prior to or during the mammalencountering the contaminated environment or item. The barrier-formingcomposition provides a barrier on the mucosa that inhibits themicroorganisms from contacting the mucosa.

In an embodiment, a method for blocking, neutralizing, or killingmicroorganisms that cause infectious disease, includes: identifying acontaminated item or environment, wherein the contaminated, item orenvironment is known, or expected to be, contaminated with harmfulviral, fungal, or bacterial microorganisms; and forming a barrier on anapparatus prior to or during the apparatus encountering the contaminatedenvironment and prior to or during the apparatus encountering a mucosaof a mammal. The barrier-forming composition provides a barrier on theapparatus that traps and kills the microorganisms, thereby preventingthe microorganisms from contacting the mucosa or causing infection.

In an embodiment, a method for blocking, killing, or neutralizingmicroorganisms that cause infectious disease in an immunocompromisedmammal, includes: identifying a disrupted area in a mucosa of themammal; and administering a therapeutically effective amount of abarrier-forming composition to at least the disrupted area of the mucosaof the mammal. The barrier-forming composition provides a barrier on thedisrupted area of the mucosa that effectively inhibits microorganismsfrom contacting a disrupted area of the mucosa.

In an embodiment, a method for blocking, neutralizing, or killingmicroorganisms introduced into a mammal's oral, nasal, or pharyngealcavity through the mammals hand-to-mouth or hand-to-nose contactincludes: identifying a contact with a contaminated item by a hand ofthe mammal, wherein the contaminated item or environment is known, orexpected to be, contaminated with harmful viral, fungal, or bacterialmicroorganisms; and administering a therapeutically effective amount ofa barrier-forming composition to an oral, nasal, or pharyngeal mucosa ofa mammal prior to the mammal's hand-to-mouth or hand-to-nose contact.The barrier-forming composition provides a barrier on the mucosa thatinhibits the microorganisms from contacting the mucosa, and neutralizesor kills the microorganisms.

In an embodiment, a method for blocking, neutralizing, or killingallergens or airborne irritants of an oral or pharyngeal mucosa in amammal prior to or during the mammal encountering a contaminatedenvironment, includes the following: identifying a contaminatedenvironment, based on the contaminated environment or item being knownor expected to be contaminated with allergens or airborne irritants tothe mucosa; administering a therapeutically effective amount of abarrier-forming composition to an oral or pharyngeal mucosa of themammal prior to or during the mammal encountering the contaminatedenvironment. The barrier-forming composition provides a barrier on themucosa that inhibits the microorganisms from contacting the mucosa.

The articles “a” and “the,” as used herein, mean “one or more” unlessthe context clearly indicates to the contrary.

The terms “item” and “apparatus” are used synonymously herein.

The term “therapeutic,” as used herein, is meant to also apply topreventative treatment.

The term “or,” as used herein, is not an exclusive or, unless thecontext clearly indicates to the contrary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of a proposed mechanism of antimicrobial activityin an embodiment of the barrier-forming composition.

FIG. 2 is a schematic showing the formation of a barrier on a mucosalsurface, as described in Example 2.

FIG. 3 is a graph showing the repair process as a percentage on awounded epithelial cell sample through cell growth and migration after 6hours to cover the scratched space in each of Examples 11-15.

FIG. 4 shows microscopic photographs showing epithelial cell growth andmigration on both untreated control Example 15 and treated Examples 16and 17 on a wounded epithelial sample.

FIG. 5 shows photos of magnified cross-sections of the barrier-formingcomposition-treated and untreated engineered human oral mucosa (EHOM) ofExamples 11-15.

FIG. 6 is graph showing an LDH assay of Examples 16-19 and 20-25.

FIG. 7 is a schema showing the method of evaluation of microbial growthin the upper and lower chambers of an EHOM assay, as described inExamples 27-28.

FIG. 8 show photographs of agar media plates showing microbial growth inthe upper and lower chambers of an EHOM assay, as described in Examples27-28.

FIG. 9 shows photographs of magnified cross-sections of thebarrier-forming composition-treated and untreated engineered human oralmucosa (EHOM) of Examples 31-32.

FIG. 10 shows photographs of microbial growth on untreated EHOM or EHOMtreated with an example barrier-forming composition, followed byinfection with C. albicans, as described in Examples 33-40.

FIG. 11 shows photographs of microbial growth on untreated EHOM or EHOMtreated with formulations followed by infection with S. mutans, asdescribed in Examples 33-40.

FIG. 12 shows photographs of microbial growth from “flow-through” media(collected from the lower chamber) of EHOM treated with an examplebarrier-forming composition, as described in Example 33-40.

FIG. 13 presents graphs showing LDH release by EHOM treated with saline(control) or example barrier-forming compositions, followed by infectionwith (A) C. albicans or (B) S. mutans, as described in Examples 40-47.

FIG. 14 is a graph showing post-antimicrobial effect of barrier-formingcompositions against bacteria and fungi, as described in Examples 48-61and 61-69.

FIG. 15 shows scanning electron micrographs of S. sanguis, C. albicans,and S. mutans, untreated or treated with barrier-forming composition, asdescribed in Examples 71-76.

FIG. 16 presents graphs depicting activity of an example barrier-formingcomposition against biofilms formed by bacteria and fungi, as describedin Examples 77-79.

FIG. 17 is a graph showing activity of an example barrier-formingcomposition on microbial biofilms after a 1-min exposure, as describedin Examples 80-81.

FIG. 18 presents fluorescent microscopy photographs showing the effectof an example barrier-forming composition on cytopathic effects (CPE) ofinfluenza (H1N1)-infected MDCK cells, as described in Examples 85-86.

FIG. 19 presents fluorescent microscopy photographs showing the effectof an example barrier-forming composition on against H1N1 virus, asdescribed in Examples 85-86.

FIG. 20 is a graph showing levels of influenza virus in infectedbarrier-forming composition treated and -untreated cells, as determinedby quantitative PCR, as described in Examples 87-88.

FIG. 21 is a graph showing direct antiviral activity of examplebarrier-forming compositions prepared with or without preservatives andantimicrobial agent (CPC) against influenza virus, determined usingquantitative PCR, as described in Examples 89-91.

FIG. 22 shows the activity of an example barrier-forming compositionagainst H1N1 virus over a 6 hour time period. Panel (A) is a graphshowing a percent inhibition in viral growth compared to an untreatedcontrol. Panels (B) and (C) are micrographs of (B) untreated and (C)barrier-forming composition treated cells.

FIG. 23 is a graph showing the activity of formulations against HIV, asdescribed in Examples 94-96.

FIG. 24 is a Western blot showing activity of Example 8 againstEpstein-Barr Virus (EBV), as described in Example 97.

FIG. 25 is a graph showing LDH levels as an indicator of cellularintegrity in untreated (control) EHOM or EHOMs tissues exposed toExamples 5-7, as described in Examples 154-159.

FIG. 26 shows representative photographs of a wounded oral epithelialcell culture treated with Example 3 (5% dilution) for 10 minutes,immediately after the wound (panel A), after about 6 hours (panel D),and after about 24 hours (panel E), as described in Example 160. PanelsB and C show an equivalent wound on an untreated control confluentculture of oral epithelial cells after about 6 hours and about 24 hours,respectively.

FIG. 27 are photographs demonstrating the ability of an examplebarrier-forming composition to coat the oral mucosal surface.

FIG. 28 are photographs showing time-lapse microscopy of bacterialgrowth after a 1 minute exposure to an example barrier-formingcomposition, as described in Examples 162-163. Images representbacterial growth after 20 min, 120 min, or 360 min post-exposure.

FIG. 29 is a graph showing the effect of a single dose of an examplebarrier-forming composition on oral microbial burden of a healthyindividual, as described in Example 164-166. (A)—Microbial load in CFUs,(B) reduction in microbial load (%) compared to baseline.

FIG. 30 is a graph showing the effect of an example barrier-formingcomposition on levels of oral microbes over a 5-day period in threehealthy adults, as described in Examples 167-169.

FIG. 31 is a graph showing the effect of an example barrier-formingcomposition on microbial burden of the oral cavity after 5-day usage in31 healthy subjects, as described in Examples 170-198.

FIG. 32 is a graph showing the microbial load in oral samples obtainedfrom three representative study participants, as described in Examples170-198.

FIG. 33 shows is a schema describing the in vitro filter insert-basedmodel to evaluate penetration of microbes across the barrier formed byexample barrier-forming compositions, as described in Examples 199-205.

DETAILED DESCRIPTION

The mucosa lining the mouth, gut, and body cavities of mammalsrepresents the first barrier to the entry of pathogenic microorganismsto mammalian bodies where they can cause both local and systemicinfections. The epithelium mucosal lining forms a barrier that reducesthe entry of commensals organisms (Monica Boirivanta and Warren Strober,“The Mechanism of Action of Probiotics” Current Opinion inGastroenterology 2007, 23:679-692).

In this application, a method and composition is disclosed that blocksor neutralizes microorganisms that cause infectious disease fromcontacting or infecting mucosa, which in turn prevents microorganismsfrom disseminating into body and causing infection. The method andcomposition incorporates an antimicrobial agent that can inhibitmicroorganisms (bacteria, fungi, and viruses) known to cause infections.The method protects human mucosa by forming a barrier over it and anantimicrobial agent is included that can kill or inhibit microorganisms(bacteria, fungi and viruses). This dual action composition and method(barrier plus antimicrobial) is applicable to human or other mammalmucosa or, for example, surfaces in the oral cavity, nasal cavity,vaginal cavity, throat, and other orifices, including, but not limitedto, the ears. It can also be applied to medical devices, such as tracheadevices. This unique and unexpected solution addresses a long-felt butunresolved need for preventing communicable diseases caused bymicroorganisms.

A barrier-forming composition that is safe (i.e. does not cause damageto the mucosa) and forms a barrier that inhibits the passage ofpathogenic microbes through the mucosal tissues is desirable. Anotherdesirable property is an ability to inhibit microbial growth throughstatic or cidal activity for an extended period of time. Without beingbound by theory, the mechanism of action of the barrier-formingcomposition disclosed herein is based on a synergistic dual-actionmechanism, in which germs are trapped in the formed barrier, andsubsequently killed by the antimicrobial active ingredient. In anembodiment the barrier-forming composition is not hydrophilic, which,without being bound by theory, is theorized to enhance it sustainedeffectiveness.

As shown in the Examples below, the properties of the barrier-formingcomposition and its effectiveness to prevent a wide variety ofcommunicable diseases were assessed using at least ten differentapproaches based on: (1) an in vitro anti-microbial susceptibilitytesting; (2) an in vitro time kill assay; (3) an in vitro biofilm model;(4) an in vitro filter insert-based model, (5) an in vivo-likeengineered human oral mucosa (EHOM) model; (6) electron microscopyevaluation; (7) hydrophobicity assay; (8) physico-chemical compatibilityassays; (9) cell culture-based model using monolayer of human celllines; and (10) human clinical trials.

The method and composition described herein may be particularly usefulwhen a human, or more generally, a mammal, has a disrupted mucosa. Adisruption may be caused be a wound or scratch. The mucosa of the oralcavity and gastrointestinal (GI) tract serve as an important mechanicalbarrier that helps to prevent a local or systemic invasion of variousmicrobes and the absorption of microbial products that are normallypresent in the oral cavity and the lumen of the gut. “Gastrointestinalmucosal injury in experimental models of shock, trauma, and sepsis,”Crit. Care Med. 1991; 19:627-41.). Derangement in the barrier functionof the mucosa plays a central role in the pathophysiology of systemicinfection. In other words, disruption of this mucosa will lead toinfections.

Elimination or reduction of the risk of a breach in the first line ofdefense is important, and the maintenance of mucosal integrity isimportant. (Anders Heimdahl, “Prevention and Management of OralInfections in Cancer Patients” Supportive Care in Cancer, Vol. 7, No. 4,224-228 (1999).) Thus, having an intact mucosa is an important hostdefense against systemic infection, particularly in immunocompromisedpatients (e.g. cancer patients). (Shahab A. Khan, John R. Wingard,“Infection and Mucosal Injury,” Cancer Treatment Journal of the NationalCancer Institute, Monographs No. 29 (2001). A barrier-formingcomposition that blocks and kills harmful microorganisms and that doesnot interfere with healing of a disrupted mucosa is a unique andunexpected solution to the susceptibility of the problems of those withdisrupted mucosa, particularly those that also have immunodeficiency.

In an embodiment, a barrier-forming composition may be administered in amethod for preventing or inhibiting an infectious disease in a mammal.By prevention, it is not meant that no infection from microorganisms ispossible, but that the risk of infection from microorganisms encounteredsubsequent to application of the barrier-forming composition is reduced.For the full preventive effect, the barrier-forming composition shouldbe applied prior to the mammal encountering a contaminated environmentor item. This is not to say that some benefit could not be obtained fromadministering the barrier-forming composition during the encounter witha contaminated environment or item. The use of the term “mammal” herein,means a human or animal commonly defined as a mammal.

In another embodiment, a barrier-forming composition is administered ina method preventing an infectious disease in mammal with a disruptedmucosa, such as for example an immunocompromised mammal. The disruptedarea in a mucosa of the mammal is identified and a therapeuticallyeffective amount of a barrier-forming composition is administered to atleast the disrupted area of the mucosa of the mammal. Thebarrier-forming composition provides a barrier on the disrupted area ofthe mucosa that effectively inhibits microorganisms from disseminatingto a disrupted area of the mucosa.

In another embodiment, the barrier-forming composition is administeredon an item or apparatus prior to the apparatus encountering acontaminated environment and prior to the apparatus encountering amucosa of a mammal. The barrier-forming composition provides a barrieron the apparatus that traps and kills the microorganisms, therebypreventing the microorganisms from passing to the mucosa or causinginfection.

In an embodiment of the method of preventing an infectious disease, astep includes identifying a contaminated environment that the mammal oritem is expected to encounter. The contaminated environment is anenvironment such as an indoor or outdoor space or a proximity to anothermammal or human that is known or expected to be contaminated withharmful viral, fungal, or bacterial microorganisms. The determination ofwhether a given environment may be contaminated may be based on the timeof year, published information on flourishing diseases in the community,or observing others that appear to be sick or spreading germs bysneezing, etc.

Predicting or identifying whether the contaminated environment or itemwill be encountered can be a decision based on whether the mammal plansor expects to enter the environment or encounter the item in the nearfuture. This may include estimating a time when the contaminatedenvironment or item will be encountered. The barrier-forming compositionmay then be administered about twenty-four hours or less prior to theestimated time of encounter with the contaminated environment or item,such as, for example, about sixteen hours or less, about twelve hours orless, about six hours or less, or about two hour or less. Thebarrier-forming composition sets up quickly and should be operable toprevent or inhibit harmful microorganisms from infecting mucosa, forexample, within less than one minute of application, such as within 30seconds. Thus, it could be applied during the encounter with thecontaminated environment or item and have effectiveness.

Harmful microorganisms are those known to cause infectious disease suchas, for example, the treatment and prevention of infectious diseases,such as communicable diseases caused by microorganisms, such as Candidaspecies (e.g. C. albicans, C. glabrata, C. krusei, C. tropicalis),Staphylococcus species (including methicillin-resistant S. aureus,MRSA), Streptococcus species (e.g. S. sanguis, S. oralis, S. mitis, S.salivarius, S. gordonii, S. pneumoniae), Acinetobacter baumannii,Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, andother microorganisms such as microorganisms that cause upper respiratoryinfections, and cold and influenza viruses. In an embodiment, thebarrier-forming composition and method of treatment and preventiondescribed herein may be useful, for example, for prevention of sexuallytransmitted diseases, such as, for example, infections caused by humanimmunodeficiency virus (HIV), Herpes simplex, or human papilloma virus(HPV), common cold (e.g. caused by rhinovirus), and infections caused byEpstein-Barr Virus (EBV).

The barrier-forming composition has shown effectiveness againstmicroorganisms with a diameter of, for example, about 30 nm or greater,such as about 100 nm (HIV, spherical), about 100 to about 300 nm(influenza, spherical and elongated forms), about 120 nm to about 260 nm(EBV spherical/disk forms), and about 30 nm (rhinovirus, spherical).Thus, the composition should also be effective against othermicroorganisms with diameters of about 30 nm, or greater than about 30nm.

The microorganisms may be air-borne microorganisms. In an embodiment themicroorganisms are those that cause communicable diseases. In anembodiment, the microorganisms do not include those that cause allergicreactions or dental problems, such as, for example, cavities (caries),gingivitis, or seasonal allergies. Similarly, in an embodiment, themethod of prevention does not solely or additionally prevent dentalproblems or allergic reactions, such as, for example, cavities (caries),gingivitis, or seasonal allergies. In another embodiment, however,microorganisms, such as fungi that may generally be classified asallergens, other allergens, and airborne irritants to the mucosa, areblocked by the barrier and the method. In the allergen blockingembodiment, the identification of the contaminated environment may, forexample, be based on the season of the year, or pollen or other allergenor irritant forecasts. It may also be based, for example, on themammal's expectation to be in a location known or expected to produce ahigh number of allergens or airborne irritants, such as, for example, anoutdoor environment, including, for example, a forest, a park, or alake.

In an embodiment, the barrier-forming composition and method oftreatment and prevention described herein may be useful, for example,for prevention of infections in environments such as hospitals andinfections common in such environments that are contaminated withinfectious microorganisms. As mentioned above, the methods andcompositions disclosed herein may be especially applicable forimmunocompromised patients. In addition, the barrier-forming compositionmay be useful for prevention of infections by microorganisms thatcommonly infect wounds.

The contaminated environment may include, for example, a publictransportation vehicle, a public gathering place, and a room or vehiclecontaining a mammal known or expected to be ill, or a close proximity toa mammal known or expected to be ill. More information on environmentscommonly recognized as contaminated environments, such as an airplane, anursery, and a health center, is disclosed in Yang, et al.,“Concentrations and Size Distributions of Airborne Influenza A VirusesMeasured Indoors at a Health Centre, a Day-Care Centre, and onAeroplanes,” J.R. Soc. Interface (Feb. 7, 2011), which is incorporatedherein by reference.

More specifically, in an embodiment, the public transportation vehiclemay be, for example, an airplane, a bus, or a taxi. A public gatheringplace may be, for example, a doctor's office, a hospital, a school, anursery, a church, a hotel, or a restaurant. The close proximity to amammal known or expected to be ill may be, for example, within a onefoot radius, or in the same motor vehicle with the mammal. A publiclyused airplane may be mentioned as a common and particularly noteworthyexample of an environment that many would identify as being acontaminated environment.

In an embodiment, the barrier-forming composition and method oftreatment and prevention described herein may be useful, for example,for prevention of infections from items that may be contaminated inactivity related treatments, such as, for example, ventilator use (whichwould include medical devices related to the ventilator and contactingthe patient). As another example of a contaminated item, treatment andprevention of fungal infections through applications to the body, and oritems or surfaces coming into contact with the body, such as shoes, mayalso be mentioned. In an embodiment the contaminated item may be, forexample, a food, a drink, utensils, drink containers and accessories, anitem for use by children, a medical apparatus, or a dental apparatus.

In an embodiment of the method of preventing an infectious disease, astep includes administering a therapeutically effective amount of abarrier-forming composition to a mucosa of the mammal prior to themammal encountering the contaminated environment or item. By atherapeutically effective amount, it is meant enough to coat thetargeted mucosa with enough of the barrier-forming composition to form abarrier that will result in a barrier layer forming on the mucosa. Forexample, about 100 microliters to about 10 ml, such as, for example,about 1 ml to about 8 ml, or about 2 ml to about 5 ml for a mouthwashformulation, or about 0.125 ml to about 2 ml, such as about 0.5 ml toabout 1 ml for a spray formulation. The dosage amount may also beexpressed in terms of a volume per square cm, such as, for example, fromabout 0.5 to about 50 μl/cm², such as, about 5 to about 40 μl/cm², orabout 10 to about 25 μl/cm² for a mouthwash formulation; or for a sprayformulation, for example, about 0.625 to about 10 μl/cm², such as, about2.5 to about 5 μl/cm². Other delivery mediums, such as dissolvablestrips, may have dosages derived from these ranges given the adjustmentsfor concentrations and other factors known to those of skill in the art.In addition, the average thickness of the film formed on the mucosa fromthe barrier-forming composition may range, for example, from about 0.001to about 0.2 mm, such as about 0.01 mm to about 0.1, or about 0.08 toabout 0.15 mm. For example, for a given human or animal, thetherapeutically effective amount can be determined based on the age orweight or size of the mammal to be treated, and the dosage may be thoselisted above. For non-human mammals, in particular, the dosage amountmay be adjusted according to the per square cm values given above andthe approximate surface area of the mucosal surface or body cavity to betreated.

In an embodiment, the barrier-forming composition administered in atherapeutically effective amount to a mucosa provides a barrier layer onthe mucosa that inhibits the microorganisms from penetrating to themucosa. In an embodiment, the inhibition of the microorganisms alsoincludes killing or deactivating the microorganism's harmful activity.In an embodiment, the barrier-forming composition blocks and/or killsall harmful microorganisms contacting the barrier-forming composition.In another embodiment, the barrier substantially blocks and/or killsenough harmful microorganisms to prevent them from causing an infectiousdisease. In the latter case, if the harmful microorganism's penetrationof the mucosa is slowed and/or diluted it will enhance the body's ownability to prevent the microorganisms from causing disease or widespreadinfection. In vitro testing demonstrates that embodiments of thebarrier-forming composition prevent all bacteria from reaching themucosal surface for long periods, including about six hours or more,about sixteen hours or more, and about twenty-four hours or more. Invitro testing shows that in viruses exposed to embodiments of thebarrier-forming composition, growth may be inhibited for about two ormore days (such as influenza), to about nine days, (such as HIV), afterwhich the viral count is still below the MIC for extended periods, suchas about two or three days. Inhibitory activity against influenza viruswas observed for up to 48 hours.

In vivo testing indicates that embodiments of the barrier-formingcomposition are therapeutically effective to reduce microbial count inthe oral cavity for about six hours or more.

In an embodiment, in a continued dosage method of prevention ortreatment, the barrier-forming composition may be administered in aseries of doses, such as, for example, about every 1 to 12 hours, aboutevery 2 to 8 hours, or about every 4 to 6 hours. This method ofprevention can be continued, for example, for a day or more, such as forabout two days to about a week. This continued dosage method may bepreferred when the subject is in prolonged contact with a contaminatedenvironment or item. In vivo testing has shown that about 80% of humansfollowing the continued dosage method show a decrease of about 50% orgreater of microbial load in the oral cavity over six days of treatment.

The mucosa, may, for example, may be a mucosal surface in the oralcavity, the nasal cavity, or the pharyngeal cavity, such as, thenasopharynx (epipharynx), the oropharynx (mesopharynx), or thelaryngopharynx (hypopharynx). The mucosa may also be in the vaginalcavity, stomach, intestine, throat or other orifices of a mammal,including, but not limited to the ear canal.

In an embodiment, administering the composition includes taking thebarrier-forming composition in a mouthwash form so as to contact theoral mucosa of the mammal. After a selected amount of time in the oralcavity, e.g. at least about 10 seconds, for example about 15 seconds toabout 5 minutes, or about 1 minute to about 3 minutes. Subsequently thecomposition is discharged from contact with the oral cavity. In anotherembodiment, the composition is administered by spraying into an oral ornasal orifice of the mammal. Other administration methods include, forexample, rubbing or applying a gelled barrier-forming composition ontothe mucosa. The barrier-forming composition may be administered to amammal through many different delivery systems, including, for example:liquids, gels, lubricants, lotions, creams, pastes, aerosolizedparticles, strips, sprays, rinses, dressings, such as for wounddressings, infusion or layering of the barrier-forming composition intoor onto products, such as on condoms, lozenges, or gums. For example,the barrier-forming composition may be administered in the form of alozenge with a liquid center comprising the barrier-forming composition,or a dissolvable strip comprising the barrier-forming composition.

In an embodiment, the barrier composition may be used to combattransmission of harmful microorganisms from hand-to-mouth orhand-to-nose contact. In this embodiment, the barrier composition isapplied to block neutralize or kill microorganisms introduced into amammal's oral, nasal, or pharyngeal cavity through the mammalshand-to-mouth or hand-to-nose contact. The method includes identifying acontact with a contaminated item by a hand of the mammal, wherein thecontaminated item or environment is known, or expected to be,contaminated with harmful viral, fungal, or bacterial microorganisms.This may include contact with the contaminated items or environmentlisted above.

After such a contact is identified, the hand or both hands that had thecontact with the contaminated item may be considered to be contaminated.At this point the barrier composition is administered in atherapeutically effective amount to an oral, nasal, or pharyngeal mucosaof a mammal prior to the mammal's hand-to-mouth or hand-to-nose contact.The barrier-forming composition then provides a barrier on the mucosathat inhibits the microorganisms from contacting the mucosa, andneutralizes or kills the microorganisms.

In an embodiment that illustrates a proposed mechanism of thebarrier-forming composition, shown in FIG. 1, the barrier-formingcomposition provides anti-viral activity. When a virus comes intocontact with a cell, it will bind to receptor on the host cell. Overtime, 5 to 6 hours, or so, the virus is internalized by the host cell,the virus multiplies inside the host cell, and it induces cell lysiscausing additional virus particles to infect other host cells.

In contrast, in a cell treated with the barrier-forming composition, aprotective barrier is on the surface of the host cell. The barrier,which is thick enough to cover the cell and any receptors on the cell,prevents the virus particle from binding to the cell receptors. Thus,infection and lysis is also prevented. The barrier-forming compositionretains the barrier for a long duration, such as a duration of about 2hours or more, a duration of about 6 hours or more, a duration of about16 hours or more, a duration of about 16 hours to about 24 hours, or aduration of about 24 hours or more, thereby protecting host cells andpreventing infection. The antimicrobial activity is also retained for along duration, such as about 2 hours or more, about 6 hours or more, orup to about 24 hours or more, thereby protecting host cells andpreventing infection.

Without being bound by theory, the same mechanism described above anddepicted in FIG. 1 is applicable to the anti-bacterial, and anti-fungalactivity of the composition and method of prevention described herein.

In an embodiment the barrier-forming composition includes a combinationof: a carbohydrate gum, a humectant; and an antimicrobial agent. In anembodiment, the composition meets the following requirements (where C isthe carbohydrate gum; H is the humectant; and A is the antimicrobialagent):

about 0.01%≤C≤about 0.4%;

about 4.5%≤H≤about 65%; and

0.050%<A

or

about 0%≤C≤about 0.4%;

about 55%≤H≤about 65%; and

0.050%<A

All percentages are by weight of the total composition.

In an embodiment, the barrier-forming composition includes glycerin orone or more similar humectant substances. The concentration of thehumectant may range from about 2% to about 70% weight percent of theentire composition, such as, for example, about 4.5% to about 65%, about7% to about 35%, or about 15% to about 45%. Humectants similar toglycerin may be classified generally as polyols. The humectants may be,for example, glycerin, sorbitol, xylitol, propylene glycol, polyethyleneglycol, and mixtures thereof. In an embodiment, glycerin may be used athigh concentrations such as about 55 to about 65% in the absence of agum.

In an embodiment, the composition also includes a gum. The gum may be,for example, a polysaccharide, xanthan gum, gum Arabic, or guar gum.Such gums may be generally classified as carbohydrate gums that have anoverall negative charge. In another embodiment, the gum may be, forexample, xanthan gum, guar gum, gum Arabic, tragacanth, gum karaya,locust bean gum, carob gum, and pectin. These gums may also be generallyclassified as carbohydrate gums that have an overall negative charge.The gum may be present in a weight percentage of the total compositionranging from about 0.01% to about 0.4%, such as for example, about 0.25%to about 0.35%, about 0.05% to about 0.25%, or about 0.4%.

In an embodiment, an antimicrobial agent is present in the composition.For example, the composition may include one or more anti-viral agents,or antifungals. In addition, the effect of such antimicrobials includesstatic and/or cidal activity.

The antimicrobial agent may include, but is not limited to cationicantimicrobial agents and pharmaceutically acceptable salts thereof,including, for example, monoquaternary ammonium compounds (QAC,cetrimide, benzalkonium chloride, cetalkonium chloride, cetylpyridiniumchloride, myristalkonium chloride, Polycide), biquaternaries andbis-biguanides (Chlorhexidine, Barquat, hibitane), and biguanides,polymeric biguanides, polyhexamethylene biguanides, Vantocil, Cosmocil,diamidines, halogen-releasing agents including chlorine- andiodine-based compounds, silver and antimicrobial compounds of silver,peracetic acid (PAA), silver sulfadiazine, phenols, bisphenols, hydrogenperoxide, hexachloroprene, halophenols, including but not limited tochloroxylenol (4-chloro-3,5-dimethylphenol; p-chloro-m-xylenol).

In addition, the antimicrobial may also be or include: antibacterialagents, both cidal and static, and different classes, for exampletetracycline, chloramphenicol, fusidic acid, fluoroquinolone, macrolideantibacterial agents, oxazolidinones, quinolone- andnaphthyridone-carboxylic acid, citral, trimethoprim and sulfamethoxazole(singly and combined), aminoglycoside, polymyxin, penicillins and theirderivatives. In addition, the antimicrobial may also include, forexample: antifungal agents in the following classes: azoles, polyenes,echinocandins, and pyrimidines. Combinations of the any of the foregoingantimicrobial agents are also contemplated. Many of the foregoing arecationic species or their pharmaceutically acceptable salts, and in anembodiment, cationic antimicrobials are utilized in the composition.

The antimicrobial may be present, for example, in an amount ranging fromabout 0.05% to 0.1% by weight of the total composition, such as, forexample, about 0.05% to about 0.6% or about 0.6% to about 0.1%. In anembodiment, the antimicrobial is about 5% or less, or about 3% or less,or about 1% or less, such as when the antimicrobial used does not causesolubility problems at higher concentrations.

In embodiments, the composition may further include other components,such as, for example, copovidone and other lubricating agents, parabenssuch as methyl paraben or propylparaben, flavoring agents,preservatives, such as sodium benzoate, buffering agents, such asmonosodium and disodium phosphate, and carboxymethylcellulose. Thesecomponents may, for example, be included in amounts ranging from about0.01% to about 5% by weight of the total composition, such as, forexample, about 0.1% to about 2%. Flavoring agents may also be used.Buffering agents (such as monosodium or disodium phosphate) may be usedto tailor the composition to the pH of the body cavity treated

Purified water may be used as the diluent component of the composition.

In an embodiment, the composition can also function to create a retainedbenefit through the inclusion of additional components providingadditional beneficial activity, such as, for example, probiotics,antacids, vitamins, drugs, nutraceuticals, silver, natural or syntheticsmall molecules, anti-oxidants, or immunostimulators, and combinationsthereof. In an embodiment, silver may be used as the antimicrobial.

Some antimicrobials, including cetyl pyridinium chloride, are known tobe negatively affected in their antimicrobial properties by additionalactive components. Thus, in an embodiment, the composition consistsessentially of the gum, the humectant, and the antimicrobial. In anembodiment, the composition is exclusive of agents for acting againstthe teeth and/or gums, including, for example, teeth whitening ordesensitizing agents. In an embodiment, the composition is alsoexclusive of cellooligosaccharides. In an embodiment, the composition isexclusive of one or more of time-release agents, allergy-reliefcompounds, azelastine, silicon based oils, essential oils, polyvinylpyrrolidone, and potassium nitrate. For the avoidance of doubt, none ofthe above should be construed to mean that all embodiments are exclusiveof these compounds.

In general, the dual-action mechanism of providing a barrier frommicroorganisms to the mucosa and an antimicrobial agent provides along-lasting effect, characterized by both in vitro. Simulated in vivo,and in vivo examples below. In in vivo examples the barrier-formingcomposition was shown to have antimicrobial effect (cidal or static) forat least 6 hours, while the barrier property was not tested in actualhuman tests, simulated in vivo tests (on artificial human mucosa EHOMs)indicated the barrier itself had a significantly extended duration past6 hours, such as greater than about 8 hours, about 6 to about 16 hours,and about 24 hours, or more. In addition, in vitro tests indicate theantimicrobial effect had a significantly extended duration past 6 hours,depending on the microorganism tested, such as greater than about 8hours, about 6 to about 16 hours, and about 24 hours, or more.

Post antimicrobial effect (PAE) is defined as suppression of microbialgrowth that persists after limited exposure to an antimicrobial agent.Having a longer PAE is considered advantageous for antimicrobial agentsas it allows for persistent inhibition of microbial growth, and mayaffect dosing regimens as agents with long PAEs may need less frequentadministration than those with short PAEs.

In embodiments of the method and composition disclosed herein the PAE ofthe composition when applied to a mucosa has a PAE that persists forabout 6 hours or more, such as about 6 hours to about 16 hours, or about16 hours to about 24 hours.

In an embodiment, the composition has a Weybridge viscosity of about 16to about 20 cps, such as, for example, about 17 to about 19 cps.

In an embodiment, at least a portion of the composition is ingested andis safe for human consumption in the therapeutically effective dosage.

It should be noted that not all mucosa in the treated cavity (e.g. oral,nasal, pharyngeal, or other) need to be covered with the barrier-formingcomposition, in order for the composition and method to be effective. Insuch a case, the composition and method are still effective to reducemicrobial load in the cavity. Without being bound to theory, due to thetrapping and killing dual-action mechanism, the barrier-formingcomposition will trap and kill microbes that otherwise would pass overthe composition to reach any uncovered portions of the cavity in whichthe composition is applied. That said, the applied composition should beeffective to cover a substantial percentage of the mucosal surface inthe treated cavity, such as, for example, about 50% or more of thecavity, such as about 75% or more, or about 90% or more.

Without being bound by theory, the barrier-forming composition is nothydrophilic which allows the barrier-forming composition to have agreater affinity to adhere to and cover the mucosal surface.Furthermore, in an embodiment, the antimicrobial being embedded in thenon-hydrophilic composition will allow for sustained antimicrobialactivity in the mucosal environment. In an embodiment thebarrier-forming composition is amphiphilic or has amphiphiliccomponents.

One measure of hydrophilicity is the Rf (relative front) value,determined by chromatography in water. In an embodiment, the compositionhas an Rf value in water of 0 to about 0.25, such as about 0.0001 toabout 0.15, or about 0.03 to about 0.1.

In an embodiment, the composition has a pH of about 4 to about 8, suchas about 5 to about 7, or about 6 to about 7.5. The pH can be tailoredto be compatible with the mucosa to be treated.

As the Examples below show, the barrier-forming composition has beenshown to block the passage of a wide variety of representative bacteriaand viruses. Because viruses are amongst the smallest infectiousmicroorganisms, and because the barrier-forming composition forms amechanical barrier blocking viruses from the mucosal cells, it isexpected that the barrier-forming composition would be an effectivepreventative treatment not only for viruses but also for largermicroorganisms, including a wide range of bacteria and fungi.

Several experiments were performed to assess the safety of thecomposition on mammals and the ability of the spray formulation to forma protective barrier on an Engineered Human Oral Mucosa (EHOM) model.The experimental evidence showed that the composition formed a barrierover tissues, which prevents microorganisms from penetrating into thetissues

EXAMPLES Example 1

Human Gingival Epithelial Cell and Fibroblast Cultures

Normal human gingival cells (epithelial cells and fibroblasts) wereobtained from ScienCell Research Laboratories (Carlsbad, Calif., USA).The fibroblasts were cultured in Dulbecco's modified Eagle's medium(DME, Invitrogen Life Technologies, Burlington, ON, Canada) supplementedwith fetal bovine serum (FBS, Gibco, Burlington, ON, Canada) to a finalconcentration of 10%. The epithelial cells were cultured in Dulbecco'smodified Eagle's (DME)-Ham's F12 (3:1) (DMEH) with 5 μg/mL of humantransferrin, 2 nM 3,3′,5′ of tri-iodo-L-thyronine.

0.4 μg/mL of hydrocortisone, 10 ng/mL of epidermal growth factor,penicillin and streptomycin, and 10% FBS (final concentration). Themedium was changed once a day for epithelial cells and three times aweek for fibroblasts. When the cultures reached 90% confluency, thecells were detached from the flasks using a 0.05% trypsin-0.1%ethylenediaminetetra acetic acid (EDTA) solution, washed twice, andresuspended in DMEM (for the fibroblasts) or DMEH-supplemented medium(for the epithelial cells).

Example 2

Engineered Human Oral Mucosa (EHOM) Tissue

The EHOM model was produced by using the gingival fibroblasts andepithelial cells of Example 1 that were used to form a complexthree-dimensional spatial cellular organization similar to that found innormal human oral mucosa. The lamina propria was produced by mixing TypeI collagen (Gibco-Invitrogen, Burlington, ON, Canada) with gingivalfibroblasts, followed by culture in 10% FBS-supplemented medium for fourdays. The lamina propria was then seeded with gingival epithelial cellsto obtain the EHOM. The tissue specimens were grown under submergedconditions until the total surface of the lamina propria was coveredwith epithelial cells. To produce stratified epithelium, the EHOM wasraised to an air-liquid interface for four more days to facilitate theorganization of the epithelium into its different strata.

The lamina propria is a thin layer of loose connective tissue that liesbeneath the epithelium and together with the epithelium constitutes themucosa. FIG. 2 shows an illustration of the EHOM mucosal tissue, with anarrow pointing to its location in a schema depicting mucosa covered withthe barrier-forming composition.

Examples 3-9

Examples of the barrier-forming compositions were created by adding theingredients listed below in a 50-mL centrifuge tube, and vortexing tobring to “free-flow” consistency. The constituents of the compositionsand their approximate amounts are given in Table I (the values in TableI are percentages by weight of the total composition):

TABLE II Example Example Example 5 Example 6 Example Example Example 3 4(control) (control) 7 8 9 Glycerin 7 35 35 35 35 7 7 Xanthan Gum 0.010.4 0.4 0.4 0.4 0.01 0.01 Cetyl Pyridinium 0.05 0.05 0.1 0.06 0.05Chloride Preservatives No No No Yes Yes Yes Yes *Purified watercomprised the remaining portion of the composition. **Preservativesincluded methylparaben (0.1%), propylparaben (0.1%), sodium benzoate(0.5%)

Based on the results below, the preservatives were found to besuperfluous to the barrier formation and antimicrobial activity.

Example 10

Monolayer Wound Repair Assay

Wound repair assays were performed on the epithelial cells andfibroblasts of Example 1. Briefly, gingival epithelial cells (1×10⁴cells) and fibroblasts (1×10³ cells) were seeded into wells of 6-wellplates and grown in Dulbecco's modified Eagle's (DME)-Ham's F12 (3:1)(DMEH) with 5 μg/mL of human transferrin, 2 nM 3,3′,5′ oftri-iodo-L-thyronine, 0.4 μg/mL of hydrocortisone, 10 ng/mL of epidermalgrowth factor, penicillin and streptomycin, and 10% FBS (finalconcentration). Upon confluency, wounds were made in the confluentmonolayer of each well using a 200 μL pipette tip.

Examples 11-15

In Examples 11 and 12, the epithelial cell cultures from Example 10 wereexposed to diluted barrier-forming compositions of Examples 3 and 4 forabout 2 minutes. In Examples 13 and 14, the fibroblast cultures fromExample 10 were exposed to diluted barrier-forming compositions ofExamples 3 and 4 for about 2 minutes. Prior to exposure, Example 3 wasdiluted with saline to a 1% concentration and Example 4 was diluted withsaline to a 5% concentration. Following exposure, the spray was washedout twice with warm sterile culture medium, then cell cultures were overlayered with DME for fibroblasts and DMEH for epithelial cells, andcultured for 6 and 24 hours in a 5% CO₂ humid atmosphere at 37° C.Control Example 15, which was an untreated culture from Example 10, wasalso tested.

Wound repair/cell migration was ascertained using an optical microscope,and digital photographs were taken (Nikon, Coolpix 950). The percentagesof wound closure (cell growth/migration) were calculated by comparingrelative wound areas before and after exposure to our barrier sprayusing formula I:

$\begin{matrix}{\frac{\begin{matrix}{\left( {{initial}\mspace{14mu} {scratch}\mspace{14mu} {size}} \right) -} \\\left( {{size}\mspace{14mu} {after}\mspace{14mu} a\mspace{14mu} {specified}\mspace{14mu} {culture}\mspace{14mu} {period}} \right)\end{matrix}}{{value}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} \left( {{inital}\mspace{14mu} {scratch}\mspace{14mu} {size}} \right) \times 100}.} & I\end{matrix}$

FIG. 3 shows a graph of the wound repair data generated from formula Iafter 6 hours post-treatment. FIG. 4 also shows photographs indicatingthe wound repair of the epithelial cells cultures treated with the 1%diluted barrier-forming composition and the control Example 15.

Following contact with the barrier-forming compositions, epithelialcells (FIG. 4A) and fibroblasts (data not shown) migrated progressivelystarting at 6 hours post contact with the barrier-forming compositionsof Examples 11 and 12. Epithelial cells were small and cuboidal inshape, in both non-exposed and barrier-forming composition-exposedcultures (FIG. 4A-E). Fibroblasts were elongated cells with smallnucleus in both culture conditions (unexposed and cetylpyridiniumchloride-based products exposed cells) (data not shown). Cell growth andmigration to cover the wounded zone were comparable in both the controlExample 15 and the treated Examples 11 and 12 as compared to theoriginal wound (FIG. 4F). Follow up over 24 hours showed that each ofExamples 11 and 12 covered the edges of the scratch giving confluentmonolayers (FIG. 4D-E).

This experiment was repeated five separate times with similar results.The treated Examples 11 and 12 did not show a major side effect ongingival epithelial cells growth/migration nor on cells differentiation(no cells presenting large cytoplasm and large nucleus).

Examples 16-19

Cytotoxicity Assay

The engineered human oral mucosa (EHOM) model of Example 2 was used todetermine whether the composition of Examples 10 and 11 were safe anddid not promote tissue damage or cell necrosis. In Example 16 and 17,the epithelium surface (10 cm²) was over layered with a thin layer (300μl) of the Example 3 barrier-forming composition at a dilution of 5% andthe Example 4 composition at a dilution of 1% (both were diluted inserum free culture medium) for time periods of about 2 minutes. Thevariation in time period was not deemed to have an effect on the resultsand just reflects the time it took to conduct the procedures. ControlExample 18 was a control that was not treated with the barrier-formingcomposition. The EHOM tissues were then rinsed twice with warm sterileculture medium and incubated in a 5% CO₂ humid atmosphere at 37° C. for24 hours. Following this incubation period, to assess whether theengineered tissue was damaged, each EHOM was macroscopically examinedfor the presence of holes due to the contact with the barrier sprayformulation. Photos were taken of these EHOM to confirm suchpossibility. Additionally, biopsies were collected from each EHOM andsubjected to histological staining using eosin and hematoxylin.

In Examples 16 and 17, EHOM exposed to Examples 3 and 4 in the 1 and 5%dilutions, similar to untreated control Example 18, do not show anymacroscopic damage such as holes. (See FIG. 5). The absence of suchdamage was monitored after 10 minute tissue contact with 5% dilutionExample 3 mouthwash (Example 7A) and 1% dilution spray (Example 7B)compositions. This was confirmed by histological analyses showingmultilayer organisation of the epithelium on the top of afibroblast-populated connective tissue. No specific damage was observedin EHOM tissues in Examples 16 or 17. The Example 16 and 17 treated-EHOMstructures were comparable to the untreated control Example 18.

The same observation was noted in an Example 19, which was the same asExample 16, except it included an EHOM treated with a 1% dilution (datanot shown).

FIG. 5 shows the effect of the barrier-forming composition on EHOMmacroscopic shape and structure. Panels A and B show control Example 18.Panels C and D show Example 16 (spray composition) and panels E and Fshow Example 17 (mouthwash composition).

Examples 20-25

Side effects, if any, of the barrier spray composition on EHOM injurywas also assessed by measuring the leakage of intracellular LDH into theculture medium.

In Examples 20-23, EHOM tissues were exposed to (A) 1% or (B) 5%dilutions of Examples 3 and 4, respectively, for 10 minutes, followed bygrowth in culture medium for 24 hours. Examples 24 and 25 were controlsthat were not treated. 50 μl of a supernatant of each of Examples 20-25were then transferred to a sterile 96-well flat-bottom plate. Each wellwas supplemented with 50 μl of reconstituted substrate mix, and theplate was incubated for 30 min at room temperature in the dark. Toestimate LDH levels, aliquots of the culture supernatant were collectedand subjected to an LDH cytotoxicity assay (Promega, Madison, Wis.,USA), as per the manufacturer's protocol. This assay measures theconversion of L-lactate and NAD to pyruvate and NADH by the releasedLDH. To stop the reaction, 50 μl of stop solution was added to eachwell. Next 100 μl of the mixture were transferred to a 96-wellflat-bottom plate, and the absorbance was read at 490 nm with an X-Markmicroplate spectrophotometer (Bio-Rad, Mississauga, ON, Canada).

In the LDH and wound repair experiments, the following test methods wereused. Each experiment was performed at least four times, withexperimental values expressed as means±SD. The statistical significanceof the differences between the control (absence of barrier spray) andthe test (presence of spray) values was determined by one-way ANOVA.Posteriori comparisons were done using Tukey's method. Normality andvariance assumptions were verified using the Shapiro-Wilk test and theBrown and Forsythe test, respectively. All of the assumptions werefulfilled. P values were declared significant at ≤0.05. Data wereanalyzed using the SAS statistical package (version 8.2, SAS InstituteInc., Cary, N.C., USA).

Results presented in FIG. 6 showed no significant difference in LDHlevels in barrier-forming composition treated structures as compared tountreated tissues. These observations were made at 6 hours (data notshown) and 24 hours (FIG. 6) post-exposure to the barrier-formingcomposition treated Examples. FIG. 6(A) shows Control Example 24 and the1% dilution treated Examples 20 and 21; and FIG. 6(B) shows a ControlExample 25, and the 5% dilution treated Examples 22 and 23. This dataconfirm the non-toxic effect of Examples 3 and 4. Similarly, contact for2 minutes with Examples 3 and 4 did not show any difference as comparedto the controls (data not shown).

Example 26

Determination Whether Barrier-Forming Composition Damages EHOMStructure.

EHOMs of Example 2 were treated with Example 4 for about 2 minutes,washed with culture medium then cultured for 24 hours. Tissue was thenexamined for possible macroscopic tissue damage (presence or not ofholes). Tissue damage was also investigated by histological analyses.For this purpose, biopsies were taken from each EHOM. They were fixedwith 4% paraformaldehyde solution and then embedded in paraffin. Thinsections (4 μm) were stained with eosin-hematoxilyn. Sections weremounted with a coverslip in 50%-glycerol mounting medium, observedthrough an optical microscope, and photographed. No damage to thetreated EHOMs was ascertained.

Examples 27 and 28

Determination Whether the Barrier-forming composition Affects MechanicalBarrier Function of EHOM Against Microbial Passage Through MucosalTissue.

In Examples 27 and 28, two approaches were used to determine whether thecontrol Examples formed a barrier that blocked the microbial passagethrough the mucosal tissues and also had an inherent anti-microbialeffect. Growth in pass-through chamber and growth on EHOM surface wasassessed by evaluating growth in agar media.

In Example 27, EHOMs of Example 2 were put in contact with 1 and 5%dilutions (diluted in serum free culture medium) of Example 4 for 2minutes. Tissues were then washed twice with serum free culture mediumthen over layered with 1×10⁶ candida microbial cells in a volume of 300μl. Tissues were then put on air-liquid culture plates and incubated for24 hours in 5% CO₂ humid atmosphere at 37° C. Next, the culture mediumunderneath the EHOM (ventral chamber) was collected and seeded onSabouraud agar plate to verify whether or not the microorganismspenetrated through the tissue and reached the culture medium below. Aculture was also obtained from the EHOM surface and seeded on Sabouraudagar plate. The process is graphically depicted in FIG. 7.

In Example 28, EHOMs of Example 2 that were treated with 1 and 5%dilutions of the Example 4 composition for 2 minutes were over layeredwith candida microbial cells for 24 hours were flipped onto Sabourauddextrose agar plates and left in place for 5 minutes. The EHOMs werethen removed and the plates were incubated for 24 hours at 30° C., afterwhich microbial growth was ascertained macroscopically and photographed.Each experiment was repeated 5 independent times with similar results.

FIG. 8 shows the results of the cultures of the EHOM surface (panels Cand D) and the culture of the pass-through liquid from the bottom(ventral) chamber (panels A and B). The A and C panels were EHOMstreated with a 1% dilution of Example 4, and the B and D panels wereEHOMS treated with a 5% dilution of Example 4. This data indicates thatExample 4 composition forms a barrier that prevents passage of microbesthrough the EHOM tissues but does not have an inherent anti-microbialeffect.

Examples 29 and 30

In Examples 29 and 30, Examples 27 and 28 were repeated, except the EHOMwere infected with S. mutans. Similar results were obtained thatindicated that the barrier-forming compositions formed a barrierpreventing the S. mutans microbes from passing through the barrier, butdid not have an antimicrobial effect.

Examples 31 and 32

Determination Whether the Barrier-forming composition Affects MechanicalBarrier Function of EHOM Against Microbial Invasion.

In Example 32, a set of EHOM tissues from Example 2 was treated with thebarrier-forming composition of Example 4 and then overlaid with C.albicans. In control Example 31 a control set was not treated with thebarrier-forming composition prior to overlayering with C. albicans.Immediately after each contact period, biopsies were taken from eachEHOM, fixed with paraformaldehyde solution, and embedded in paraffin.Thin sections (4 μm) were stained with eosin-hematoxylin. Sections wereobserved using an optical microscope to analyze the invasion/penetrationof microbial cells into the tissue. Following microscopic observations,representative photos were taken from each condition and presented. Theexperiment was repeated three times with similar results. Similarresults were also obtained with treatment with Example 3 (data notshown).

FIG. 9 shows the effect of the barrier-forming composition on microbialinvasion of EHOM tissues. Panel (A) is a representative photograph ofthe untreated control Example 31, and panel (B) is a photograph of thetreated Example 32. The arrow indicates invading fungal hyphae in theuntreated control Example 31.

Examples 33-40

The EHOM model described above was also used to evaluate the ability ofExamples 5-7 to form a barrier that: (a) prevents oral bacteria (S.mutans) and fungi (Candida albicans) from penetrating/invading humanoral mucosa, and (b) does not cause damage to host cells (cytotoxicityassay).

Examples 33-40 were formulated according to Table III below.

TABLE III Barrier-forming composition Pre- Treatment Microbe OverlayFigure reference Example 33 None C. albicans FIG. 10(A) Example 34Example 5 C. albicans FIG. 10(B) Example 35 Example 6 C. albicans FIG.10(C) Example 36 Example 7 C. albicans FIG. 10(D) Example 37 None S.mutans FIG. 11(A) Example 38 Example 5 S. mutans FIG. 11(B) Example 39Example 6 S. mutans FIG. 11(C) Example 40 Example 7 S. mutans FIG. 11(D)

In Examples 33-40, after pre-treatment and incubation according to theprocedures of Examples 27 and 28: (1) the flow-through medium wascollected from the lower chamber; and (2) tissues were flipped andplaced onto the surface of Sabouraud dextrose agar Petri dishes, andincubated for 24 hours. Collected flow-through media were spread ontoagar media plates, and incubated for 24 hours also as described inExamples 27 and 28. Table III also indicates the Figure in which a photoof each Example was taken showing the microbial growth on each flippedExample culture.

FIGS. 10 and 11 show that both Candida and Streptococcus were able togrow on the surface of EHOM treated with the compositions of Examples5-7. In contrast, as shown in FIG. 12, no microbial growth was observedwhen the “flow-through” medium collected from the lower chambers ofEHOMs of Examples 36 or 40, i.e. those treated with the Example 7composition This indicates that treatment of the EHOMs with the Example7 composition did not cause damage to the surface of the mucosal tissuesand organisms were unable to penetrate the treated EHOM. Similar resultswere obtained with EHOM treated with the compositions of Examples 5 and6 (data not shown). These data indicate that the combination ofglycerine and xanthan gum is capable of forming a protective barrier onmucosal tissues.

Examples 41-47

Tested Formulations are not Toxic and do not Cause Damage to theCells/Tissues

In Examples 41-47, the EHOM model was used to assess the toxicity of thecomposition. Examples 41-47 were formulated as stated in Table IV.

TABLE IV Barrier-forming composition Pre- Treatment Microbe OverlayFigure Reference Example 41 None C. albicans FIG. 13(A) Example 42Example 5 C. albicans FIG. 13(A) Example 43 Example 6 C. albicans FIG.13(A) Example 44 Example 7 C. albicans FIG. 13(A) Example 41A None S.mutans FIG. 13(B) Example 45 Example 5 S. mutans FIG. 13(B) Example 46Example 6 S. mutans FIG. 13(B) Example 47 Example 7 S. mutans FIG. 13(B)

After pre-treatment and incubation according to the procedures ofExamples 27 and 28, culture supernatant was collected from the Example41-48 EHOM tissues and used to measure LDH activity.

As shown in FIG. 13, no significant increase in LDH levels was observedin Examples 41-48 irrespective of whether the formulations containedcetylpyridinium chloride with or without preservatives and infected witheither Candida albicans or S. mutans, respectively. These data confirmedthe non-toxic effect of the Example barrier-forming compositions andthat these formulations maintained the integrity of the host mucosaltissues.

Data are mean±SD and were computed as stated in Example 25 above. Nosignificant difference between untreated and treated tissues was noted.

Taken together, the data indicates that the example compositionsrepresent an effective and a safe barrier that can preventmicroorganisms from penetrating and invading human mucosal tissues.

Examples 48-61

Preclinical evaluation of the barrier-forming composition showed thatthe composition was effective against many bacteria and yeasts. Theantimicrobial activities of the Example 7 barrier-forming compositionwere evaluated against a number of clinical isolates obtained frompatients, including S. salivarius, P. gingivalis, S. pyogenes, S.pneumonia, Fusobacterium nucleatum, S. mutans, S. aureus, Y.enterocolitica, S. oralis, S. mitis, C. albicans, C. krusei, C.tropicalis, and C. glabrata. Activity of the Example 7 barrier-formingcomposition was evaluated by determining its minimum inhibitoryconcentration (MIC) using reference methods described in the Clinicaland Laboratory Standards Institute (CLSI) documents M07-A8, M11-A7, andM27-A3.

A standardized inoculum of several types of aerobic or anaerobicbacteria (1×10⁴ cells/ml) was incubated with serially diluted solutionsof Example 7 (containing 0.1% CPC, or 1 μg/ml) or 2% chlorhexidinegluconate (CHX, 20 μg/mL) as a comparative example. Cells were allowedto grow in the presence or absence (growth control) of the test agentsfor 24 hours. The MIC for each agent was defined as the concentrationthat induced a 100% growth inhibition (compared to no-drug control).

A similar microdilution-based CLSI method (M27-A2) was used to evaluatethe activity of Example 7 against albicans and non-albicans Candidaspecies.

TABLE V Example 7 (μg/mL Chlorhexidine Organism CPC) (μg/mLchlorhexidine) Example 48 S. salivarius 0.98 19.6 Example 49 P.gingivalis 0.98 19.6 Example 50 S. pyogenes 0.98 19.6 Example 51 S.pneumonia 0.98 19.6 Example 52 F. nucleatum 1.95 19.6 Example 53 S.mutans 1.95 19.6 Example 54 S. aureus 3.91 19.6 Example 55 Y.enterocolitica 3.91 19.6 Example 56 S. oralis 500 19.6 Example 57 S.mitis 500 19.6 Example 58 C. albicans 0.25 19.6 Example 59 C. krusei0.06 19.6 Example 60 C. tropicalis 0.06 19.6 Example 61 C. glabrata0.125 19.6

The barrier-forming composition was also found to have potentantimicrobial activity against: MRSA, Acinetobacter baumannii,Streptococcus sanguis, S. gordonii, and Aggregatibacteractinomycetemcomitans.

As can be seen in Table V, the Example 7 composition exhibited potentactivity against many aerobic and anaerobic bacteria, as well as thefungi.

The MIC of the Example 7 barrier-forming composition against S. oralisand S. mitis was noticeably elevated (500 μg/mL) compared to otherorganisms. It is interesting to note that S. oralis and S. mitis arenormal commensals of the oral cavity. Activity of the commonly usedantimicrobial chlorhexidine (2% solution) was also determined by thesame method. Table V shows the MIC of the Example 7 barrier-formingcomposition and chlorhexidine (2% solution) as a comparative exampleagainst various microorganisms.

Taken together, these results demonstrate that Example 7 possessespotent activity against pathogenic bacteria and fungi commonly isolatedfrom the oral cavity. This activity was more potent than that observedfor chlorhexidine.

A similar activity profile was observed for the barrier-formingcompositions of Examples 10 and 11.

Example 62

As a further comparison, published data shows that the testedbarrier-forming composition has a better or at least equivalent MICcompared to CPC alone (i.e. not in a composition according to thebarrier formulation disclosed herein). See Frank-Albert Pitten and AxelKramer, “Efficacy of Cetylpyridinium Chloride Used as OropharyngealAntiseptic,” Arzneim.-Forsch./Drug Res. 51 (II), pp 588-595 (2001),which is incorporated herein by reference. The data varies based on themicroorganism tested, but, for example, CPC (alone) against S. mutanshas an MIC of 5.0-6.25 μg/mL, which is much less effective than the 1.95μg/mL reported in Example 53. This was an unexpected result since CPChas the risk of losing its activity when mixed with other excipientchemicals in a formulation. See Department of Health and Human Services(Food and Drug Administration) (1994) Oral Health Care Drug Products forOver-the-Counter Human Use; Tentative Final Monograph for OralAntiseptic Drug Products. Proposed Rules (21 CFR Part 356, Docket No.81N-033A, RIN 0905-AA06). Federal Register 59:6084-124.

Examples 63-69

Duration of Antimicrobial Activity of Barrier-Forming Compositions InVitro: Determination of Post-Antimicrobial Effect (PAE)

The PAE of Example 8 against several microorganisms was evaluated inExamples 63-68. Control Example 69 was also provided. Severalmicroorganisms were exposed to Example 8 (at a concentration equal tothe MIC) for 1 min followed by three washes to remove residualformulation. The treated cells were then spread on agar medium plates,which were incubated at 37° C., and the time taken for the cells toregrow was determined. PAE was expressed as the time (in hours) forwhich growth inhibition (%) was maintained by the Examples 63-68,compared to the untreated control Example 69.

As shown in FIG. 14, Example 8 exhibited a PAE ranging between 4 hoursto 24 hours, depending on the organism tested (S. aureus, S. pneumonia,S. gordonii, S. sanguis, S. salivarius, and S. mitis). Similar activityof Example 8 was observed against Candida (data not shown). OtherExample barrier-forming compositions exhibited similar PAE againstmicroorganisms.

Example 70

Testing of PAE for the Example 7 barrier-forming composition against S.mutans compared to a similar comparative Example with lower CPC contentshowed that the PAE of Example 7 was 24 hours, while that of ComparativeExample 70 was 6 hours. Thus demonstrating the Example 7 exhibitsgreater prolonged antimicrobial activity than comparative Example 70,and that additional amounts of CPC have more than a simple additiveeffect on anti-microbial activity.

Examples 71-76

Scanning electron microscopy was also used to show that treatment of S.sanguis, (Example 71), S. oralis, (Example 72), and C. albicans (Example73) with the composition of Example 3 resulted in destruction ofcellular integrity.

In Examples 71-73, cells were grown in the presence of Example 3 for 24hours. Next, the cells were washed to remove residual formulation,dehydrated by passing through a series of alcohol solutions (10% to100%, v/v) and processed for SEM analysis. Control Examples 74-76differed from Examples 71-73 in that they were not grown in the presenceof Example 3.

The SEM photos showed that unlike untreated control Examples 74-76,which demonstrated healthy intact cells (FIG. 15 A, C, E), microbesexposed to the Example 3 barrier-forming composition were deformed,collapsed, and exhibited total destruction of cellular integrity withclear evidence of leakage of cytoplasmic material. (FIG. 15, B, D, F).

Examples 77-79

Since biofilms are precursors to certain infectious diseases, inExamples 77-79, experiments were performed to determine whether thebarrier-forming compositions can prevent formation of biofilms bybacteria and yeasts. Biofilms were formed using an in vitro model. SeeChandra et al. “In vitro Growth and Analysis of Candida Biofilms” NatureProtocols 3(12): 1909-1924 (2008).

In Examples 77-79 a standard biofilm model was employed to determinewhether the Example 3 barrier-forming composition exhibits activityagainst bacterial and fungal biofilms. In Examples 77-79, threedifferent microorganisms (C. albicans, S. oralis, and S. salivarius)were adhered on substrate for 90 minutes to allow biofilms to form toadhesion phase. Next, discs containing the adherent bacteria wereincubated for 15, 30 or 60 minutes with 50% concentration of Example 3(1:1 dilution with appropriate medium). Following incubation, biofilmswere scraped, spread on culture media, incubated and colony formingunits (CFUs) were determined. Media diluted with phosphate bufferedsaline (PBS, 1:1) were used as a control. Table VI reports data at 0(Control), 15, 30, and 60 minutes.

TABLE VI Effect of Barrier-forming composition on Early Phase Biofilms(log CFU) Example 77 Example 78 Example 79 Exposure time C. albicans S.oralis S. salivarius Control 5.44 3.25 3.16 15 min 0 0 0 30 min 0 0 0 60min 0 0 0

FIG. 16 also reports data on Examples 77-79 as a graph of % inhibitionversus growth control. These results showed that Example 3barrier-forming composition inhibited bacterial and fungal microbes withan MIC of 0.2% against biofilms formed by S. salivarius, S. oralis, orC. albicans.

Examples 80 and 81

In Example 80 we evaluated the effect of 1 minute exposure of C.albicans early phase biofilms to Example 3, and found that even with anexposure for as short a time as 1 minute, it was able to inhibit biofilmformation (FIG. 17). Example 81 was an untreated control sample.

Examples 82-84

Ability of Barrier-Forming Composition to Treat Mature Biofilms

To determine whether the barrier-forming composition can treat biofilms,we evaluated its activity against fully formed mature biofilms. Biofilmswere grown to mature phase, and then exposed to Example 7 for 2 or 4hours, and the resulting CFUs were determined. A composition that causesat least 2-log reduction in microbial CFUs compared to untreated cellsis considered to be effective against microbial biofilms.

As shown in Table VII, exposure to Example 7 resulted in completeeradication of biofilms formed by C. albicans and S. oralis, and a3.4-log reduction in CFUs for biofilms formed by S. salivarius comparedto the untreated control (log CFU=3.95 vs. 7.36, respectively).

TABLE VII Effect of Example 7 on mature biofilms (log CFU) Example 82Example 83 Example 84 Exposure time C. albicans S. oralis S. salivariusControl 5.60 7.40 7.36 2 h 0 0 4.00 4 h 0 0 3.95

In summary, the results indicate that Example 15 possesses potentactivity against biofilms formed by bacteria and fungi.

Examples 85-86

The Barrier-forming composition is also Active Against Viruses

The activity of barrier-forming composition against viruses, includingrespiratory viruses (influenza virus H1N1, strain 2009/H1N1/infA) andthe human immunodeficiency virus (HIV) was determined.

The barrier-forming composition inhibits the infectivity of influenza A

To evaluate the effect of the barrier-forming composition on theinfectivity of influenza virus, Madin Darby canine kidney (MDCK) cellswere grown to ≥90% confluence at 37° C. prior to infection. MDCK cellsare used routinely for assays involving influenza viruses.

In Example 85 cell monolayers were exposed to the Example 7barrier-forming composition. In control Example 86 the cell layers wereexposed to optiMEM (+P/S,+Lglu) tissue culture media for differenttimes: (1) T1: 30 min exposure, (2) T2: 1 h exposure, (3) T3: 2 hexposure. Next, the formulation was removed and the cell monolayers wereinfected with influenza virus (multiplicity of infection (MOI)=0.1).Cells that were untreated or infected immediately after exposure (T0)were used as baseline controls. Infected cells were then centrifuged,resuspended in 500 μL of growth medium, and incubated at 32.5° C. for 48hours. Immunofluorescence microscopy (using FITC labeled anti-influenzaantibody) was also used to evaluate the effect of the Example 7barrier-forming composition on the ability of influenza virus to infectmammalian cells.

FIG. 18 shows the effect of Example 7 on cytopathic effects ofinfluenza-infected MDCK cells (Example 85) (panels A and C), and controlExample 86 (panels B and D). Images were obtained from: phase contrast(A-B), and immunofluorescence microscopy (C-D). No identifyingcytopathic effect (CPE) was observed in formulation-treated cells.Untreated cells displayed typical CPE including focal rounding anddegenerative changes.

The data showed that exposure of cell monolayers to Example 7 for 30minutes, 1 hour, or 2 hours remained confluent and healthy (Example 85).In contrast, in the untreated cells and cells treated immediately priorto infection (T0) (control Example 86) demonstrated substantialcytopathic effect. As seen in FIG. 18 panel C, no fluorescence wasobserved in the barrier-forming composition treated cells of Example 85,while the untreated cells of Example 86 exhibited fluorescence (FIG. 18panel D).

Further fluorescence microscopy images corresponding to Examples 85 and86 are presented in FIG. 19.

Examples 87 and 88

Activity of Barrier-forming composition on Viral Load using QuantitativePCR.

FIG. 20 shows levels of influenza virus in infected treated cells(Example 87) and untreated cells (Example 88), as determined byquantitative PCR. In Example 87, cells were treated with Example 7 andin control Example 88 the cells were left untreated. Later thesupernatants were collected and analyzed for the presence of virus.

Cell culture supernatants from the same assay as in Examples 87 and 88were collected and nucleic acid extracted using QIAamp Viral RNA Kit(QIAGEN, Valencia, Calif.). Random hexamer primers (Invitrogen Carlsbad,Calif.) were used to create a cDNA library for each specimen. Reversetranscription reactions were performed with M-MLV RT (Invitrogen,Carlsbad, Calif.) according to the manufacturer's specifications.Quantitative analysis was performed on a StepOne Plus Taqman Real TimePCR (Applied Biosystems, Branchburg, N.J.) using TaqMan Universal PCRMaster Mix (Applied Biosystems, Branchburg, N.J.), 2 μl of cDNA sample,and primers/probes targeting the influenza matrix gene. A referencestandard was prepared using a cDNA fragment of the H1N1 matrix gene andhuman RNAse P amplified by conventional RT-PCR, gel purified (QIAquick,Qiagen, Valencia, Calif.), and quantified using a spectrophotometer(Beckman Coulter, Brea, Calif.).

As shown in FIG. 20 and Table IV, the Example 87 cells treated Example 7for 30 min or 60 min did not have detectable influenza at 48 hours postinfection. Moreover, treatment with Example 7 for 2 hours resulted in a6-fold decrease in viral load, compared to the untreated control orthose treated immediately prior to infection (Example 88).

TABLE VIII Example 87 Example 88 (control) 30 min 0 192000 60 min 079800 120 min  23400 143000

Examples 89-91

Barrier-forming composition has direct antiviral effect againstinfluenza virus

To determine whether the barrier-forming composition has directantiviral activity against influenza virus, we infected African GreenMonkey Kidney (CV-1) cells (grown in 24-well plates to 90% confluence)with influenza virus that was pre-treated with Example 7. CV-1 cells areroutinely used a highly susceptible substrate for diagnosis and study ofviruses.

In Examples 89-91, a standardized amount of influenza (0.1 MOI) waspretreated for 5 minutes at room temperature with: (1) Example 7 (toform Example 89), (2) control Example 6, a compound without CPC but withpreservatives (to form Example 90), and (3) control Example 5 placeboalone (a compound without CPC and preservatives) (to form Example 91).After the 5 minute incubation virus/drug mix was diluted by anadditional equal volume with optiMEM (+P/S,+Lglu) to dilute out thetreatment compositions.

In Examples 89-91, CV-1 cells were prepared as described in above. TheExample 89-91 treated and untreated viruses were then inoculated ontothe cells as described above.

Influenza viral load was determined by real time PCR as described above.The data as shown in FIG. 21 showed significant decrease in viral loadfor influenza virus pretreated with the Example 7 barrier-formingcomposition containing the antimicrobial agent CPC (Example 89),compared to those containing only the barrier-forming composition and/orpreservative but no CPC (Examples 90 and 91). Pre-treatment of viruswith Example 7 exhibited significant decrease in viral copies, comparedto formulations with no CPC.

These results demonstrate that the Example 7 barrier-forming compositionpossesses direct antiviral activity against influenza virus that is notinherent in Examples 5 and 6.

Examples 92 and 93

In Examples 92 and 93, the barrier-forming composition's ability toinhibit the infectivity of influenza A (2009/H1N1/infA) was tested.African Green Monkey Kidney (CV-1) cells were grown in 24-well plates to90% confluence. Next, the barrier-forming composition, Example 7, wasapplied to the cells (20% Example 7, 80% OptiMeM, working CPCconcentration of 0.02%.) in Example 92. Each time point matched withcontrol Example 93 (No barrier-forming composition applied, 100%OptiMeM). The barrier-forming composition was allowed to dwell on thesurface for 30 minutes, and then removed from the ceil monolayer. Cellswere thoroughly washed twice with sterile optiMEM (+PfS,+Lglu).Influenza was inoculated at MOi=0.1 at 30 minute intervals from T0through T+6 hours. Following infection, cells were then centrifuged @2200 rpm×30 minutes and 500 μl of optiMEM (+P/S, +Lglu, 2 μg/ml trypsin(sigma-Aldrich, St Louis, Mo.)) was applied. Infected cells were grownat 32.5° C. for 96 hours at 5% CO₂. The influenza viral load wasdetermined by real time PCR.

As shown in FIG. 22, pre-treatment of host monolayers withglycerine-xanthan gum formulation results in inhibition of viralinfection by up to 84.93% compare to untreated controls. The fact thatinhibition of viral infection was observed in host cells despite removalof the barrier-forming composition demonstrates that the barrier-formingcomposition formed a protective barrier on host cells, which preventedviral invasion at least 6 hours.

FIG. 1 may be referred to as a possible mechanism accounting for theinhibition of infection.

Examples 94-96

Barrier-Forming Composition Exhibits Activity Against HIV

Examples 94-96 determined whether the barrier-forming compositionpossessed activity against HIV. Host MT mammalian cells were plated into96-well round bottom plates at a density of 15,000 cells/well inRPMI/10% FBS/PS. The next day (Day 2), virus was pretreated with controlExample 5 (to form Example 94), control Example 6 (to form Example 95),or Example 7 (to form Example 96) for 5 minutes and added to cells.After 24 hours of exposure to formulation, the MT (macaque) mammaliancells were washed 3 times with phosphate buffered saline (PBS) and freshmedia was replaced. Supernatant (10 μL) was collected post-treatment onDays 1, 2, 5, 6, 7, and 9, and the viral load was determined by reversetranscriptase (RT) activity. FIG. 23 shows a graph of the viral copiesper mL for each of Examples 72-74 over a 9 day span.

The results showed that Example 7 in Example 96 exhibited anti-HIVactivity at all time points monitored post-treatment.

The control Example 5 or control Example 6 without CPC and/orpreservative in Examples 94 and 95 exhibited only minimal anti-HIVactivity.

In summary, our findings demonstrate that the barrier-formingcomposition Example 7 containing CPC exhibits long-lasting antiviralactivity against HIV.

Example 97

Representative organisms viral lesions are important infections indifferent mucosal tissues. In Example 97 an experiment was performed todetermine whether the barrier-forming composition exhibits activityagainst the common oral Epstein-Barr virus (EBV). Western blotting wasused to evaluate the ability of the Example 8 barrier-formingcomposition to degrade lytic viral protein EAD (indicating inhibition ofviral replication).

In Examples 97, EBV-infected gastric epithelial cells were exposed todifferent dilutions (1:16, 1:32 and 1:64) of Example 8, and the presenceof EAD protein was detected using specific antibodies. Presence ofcellular β-actin was used as an indicator of epithelial cell integrity.As shown in FIG. 24, 1:64 dilution of Example 8 degraded EAD withoutaffecting cellular actin. These results demonstrate that Example 8specifically inhibits viral replication, and as such, is an effectiveanti-viral and useful for prevention of viral infection.

Examples 98-100

Duration of Anti-Microbial Barrier Versus Commercial Mouthwash Product

To determine the duration for which the barrier-forming composition canmaintain the antimicrobial activity, bacteria and fungi were exposed toan EHOM of Example 2 that was treated with the barrier-formingcomposition of Example 7 in a well and an EHOM of Example 2 that wastreated with a comparative commercial product in a well for 2 minutes.The bacterial and fungal microbes were overlaid on top of the controluntreated EHOM (Example 98) and the treated EHOMs (Example 99 andComparative Example 100). Next the residual (flow-through) solution wasremoved from the bottom well (lower chamber of the EHOM model) andspread onto agar medium plates. FIG. 7 depicts this test method forfurther clarity. These plates were then incubated at 37° C., and thenumber of microbial cells (colony forming units, CFUs) growing after 24hours were counted.

In control Example 98 an untreated EHOM was tested. In Example 99 S.mitis bacteria was overlaid on the barrier-forming composition asdescribed above. Example 100 is a comparative example showing theactivity of commercially available LISTERINE (containing ethanol(26.9%), menthol, thymol, methyl salicylate, and eucalyptol) against S.mitis bacteria. Table IX shows the results.

TABLE IX CFUs of S. mitis bacteria in flow through liquid from EHOM Timepost- Example 98 Example 100 exposure (control) Example 99 (comparative)2 hours 1150000 5820 780000 4 hours 1400000 5500 800000 6 hours 16000006000 840000

Examples 101-103

In Examples 101-103, the same procedure of Examples 98-100 was performedexcept Candida albicans fungus was tested on the barrier-formingcomposition as described above. Table X shows the results. Example 103is comparative, showing the activity of commercially availableLISTERINE.

TABLE X CFUs of Candida albicans in flow through liquid from EHOM Timepost- Example 101 Example 103 exposure (control) Example 102(comparative) 2 hours 1150000 12000 124000 4 hours 2900000 12000 2520006 hours 3900000 13000 350000

The data further showed that Example 7 barrier-forming compositionmaintained activity for up to and including 24 hours. Taken together,these results showed that unlike LISTERINE, the Example 7barrier-forming composition continued to maintain an intact barrier onEHOM tissues for up to and including 24 hours.

Examples 104-153

To identify further examples of concentrations of glycerin and xanthangum that can form a barrier effective in preventing the passage ofmicroorganisms, different concentrations of the gum xanthan gum andhumectant glycerin were tested (5%-95% glycerin; 0.005%-0.5% xanthangum) singly and in combination using an in vitro filter insert-basedbarrier model. FIG. 33 shows the general test method used for Examples104-153.

Filter inserts of 3 μm and 8 μm diameter pore size were used for testingthe passage of bacteria (Streptococcus salivarius) and fungi (Candidaalbicans), respectively. Glycerin or xanthan gum or their combinations(100 μL aliquots) were overlaid on the surface of the filter to form abarrier. The filter had a diameter of 24 mm. Thus, the film had athickness of approximately 0.01 mm on the filter, mimicking a value inthe range of thicknesses of the composition film when applied in atherapeutically effective amount to the mouth. Next, 5×10⁴ cells ofeither bacteria or fungi were applied on top of the formed barrier inthe filter inserts. Next, we placed these filter inserts on the surfaceof agar medium (Brain Heart Infusion (BHI) medium for bacteria,Sabouraud Dextrose (SD) medium for fungi) in 6-well plates. The platesalong with the filter inserts were incubated overnight for 24 hours at37° C.

The plates were monitored for the presence of bacterial or fungal growthCFUs (colony forming units) in the agar medium as well as in the filterinsert. Microbial growth in the filter insert only, but not in the agarmedium, demonstrated that an effective barrier was formed on the filter,which prevented passage of microorganisms. Conversely, growth in theagar medium around the filter insert suggested that the tested agentsfailed to form an effective barrier, allowing the organisms to gothrough the filter.

The results reported in Table XI showed that glycerin was able to form abarrier at concentrations greater than or equal to 55%, when testedalone. In contrast, xanthan gum alone did not form a barrier at any ofthe concentrations tested (ranging from 0.005% to 0.4%). However, it wasobserved that when combined with 0.01% xanthan gum, a barrier was formedat glycerin concentrations 7%, 45%, 55%, and 65%. Furthermore,combination of 0.4% xanthan gum with glycerin concentrations of 7%, 15%,25%, 35%, 45%, 55%, and 65% also formed a barrier. Therefore, specificcombinations of glycerin and xanthan gum were identified that can form abarrier that prevents passage of microorganisms in an in vitro filterinsert-based model.

TABLE XI Examples Example Examples Examples Examples Examples Examples104-112 113 114-121 122-129 130-137 138-145 146-153 Xanthan Gum (%) 00.005 0.01 0.05 0.1 0.2 0.4 Glycerine 0 No No No No No No No (%) 5 No NoNo No Yes No 7 No Yes Yes 15 No No No No Yes Yes 25 No No No No Yes Yes35 No No No No Yes Yes 45 No Yes No No Yes Yes 55 Yes Yes No No Yes Yes65 Yes Yes No No Yes Yes ‘No’: no barrier formed; ‘Yes’: barrier formed

Microbial cells retained by the compositions of Examples 104-153 formedon filter inserts were trapped by the barrier, and were viable, thusdemonstrating that the formed barrier does not have an inherentantimicrobial property without an antimicrobial agent. In other words,the microbes retained in the barrier were still active and could pose athreat to infection; for example, if they are freed from the barrier byabrasion or after the barrier loses its integrity.

Examples 154-157

In addition to the Examples above on EHOM samples, further testing wasperformed to further demonstrate the barrier-forming composition (a)does not damage the host tissues, and (b) is able to prevent microbialinvasion into the human mucosal tissues. These criteria were tested fortwo representative combinations (glycerine:xanthan gum; 7%:0.01% and35%:0.4%), selected based on the in vitro results of Examples 104-153,because they successfully formed a barrier. Formulations containingthese two combinations were tested using the EHOMs of Example 2 thatmimic the mucosal lining.

The EHOMs were treated with the various formulations of Examples 3 and 4in either 1% (Examples 154 and 155) or 5% (Examples 156 and 157)dilutions in normal saline [0.9%]) for about 2 minutes to form Examples154-157. Each EHOM was covered with 300 μL of one of the testedformulations and left under the sterile hood for 10 minutes at roomtemperature (25° C.). At the end of the contact period, tissues werewashed twice with culture medium to remove the formulations.

Example 158

After treatment with Examples 3 and 4, the EHOMs were then examined forpossible macroscopic tissue damage (presence of holes). Tissue damagewas also investigated by histological analyses. For this purpose,biopsies were taken from each EHOM and fixed with 4% paraformaldehydesolution and then embedded in paraffin. Thin sections (4 μm) werestained with eosin-hematoxilyn and mounted with a coverslip in50%-glycerine mounting medium, observed through an optical microscope,and photographed. The treated EHOMs and a control untreated EHOM wereexamined for macroscopic tissue damage and structural changes. There wasno visible damage (holes) in the untreated or treated tissues at amagnification of X500 in five different EHOMs tested.

Example 159

Evaluation of Barrier-Forming Composition Causing Damage to Host Cells(Cytotoxicity Assay).

The Example 156 and 157 EHOMs that were treated with the 5% dilution ofExamples 3-7 of the barrier-forming composition were then over-layeredwith 1×10⁶ cells of either C. albicans or S. mutans in a volume of 100μl. The EHOMs were then placed on air-liquid culture plates andincubated for 24 hours in 5% CO₂ humid atmosphere at 37° C. Followingthis incubation period, aliquots of the culture supernatant werecollected and subjected to a Lactate dehydrogenase (LDH) cytotoxicityassay (Promega, Madison, Wis., USA), as per the manufacturerinstructions. 50 μl of each supernatant were transferred to a sterile96-well flat-bottom plate. Each well was supplemented with 50 μl ofreconstituted substrate mix, and the plate was incubated for 30 minutesat room temperature in the dark. To stop the reaction, 50 μl of stopsolution was added to each well. Next 100 μl of the mixture wastransferred to a 96-well flat-bottom plate, and the absorbance was readat 490 nm with an X-Mark microplate spectrophotometer (Bio-Rad,Mississauga, ON, Canada). LDH was assessed using LDH cytotoxicity assay.Data are means±SD. No significant difference between EHOMs treated withthe Example composition and an untreated, uninfected EHOM control wasnoted. FIG. 25 graphically shows the results of these tests on anuntreated control example, and those treated with Examples 5, 6, and 7.Similar results were obtained for the EHOMs treated with Examples 3 and4. The tested Examples all maintained the integrity of the mucosa.

Example 160

Effect of Barrier-Forming Composition Formulations on Gingival CellGrowth/Migration

Wound repair assays were performed. Briefly, oral (gingival) epithelialcells (1×10⁴) and fibroblasts (1×10³) were seeded into wells of 6-wellplates and grown in appropriate culture medium. Upon confluency, woundswere made in the confluent monolayer of each well using a 200 μL pipettetip. Cultures were then exposed to 1 and 5% by weight dilutions ofExamples 3 and 4 for about 2 minutes. Following exposure, theformulations were washed out twice with warm sterile culture medium, andthen cell cultures were over layered with DEM for fibroblasts and withDEM for fibroblasts and DMEH for epithelial cells, and cultured for 6and 24 hours in a 5% CO₂ humid atmosphere at 37° C. Wound repair/cellmigration was ascertained using optical microscope, and digitalphotographs were taken (Nikon, Coolpix 950). FIG. 26 showsrepresentative photographs of a wounded oral epithelial cell culturetreated with Example 3 (5% dilution) for 10 minutes, immediately afterthe wound (panel A), after about 6 hours (panel D), and after about 24hours (panel E). Panels B and C show an equivalent wound on an untreatedcontrol confluent culture of oral epithelial cells after about 6 hoursand about 24 hours, respectively. Similar results were seen for the cellculture treated with Example 4.

The percentage of wound closure (cell growth/migration) was calculatedby comparing relative wound areas before and after exposure to theformulations using formula I stated above.

The epithelial cells were small and cuboidal in shape in both treatedand untreated cultures. Similar results were observed for scratch woundmodel using fibroblasts (data not shown). Taken together, this dataindicate that example compositions are not toxic and do not negativelyimpact cell growth/migration and wound healing.

Example 161

Glycerine-Xanthan Gum Formulations Form a Coating on the Human OralMucosa

To determine whether glycerine-xanthan gum formulation can form acoating on the human oral mucosa, we spiked the Example 7 formulationwith Gentian Violet (GV) as a marker dye. The spiked product (750 μL)was sprayed onto the oral cavity of human volunteers. Post-application,the oral cavity was inspected for staining, and the images were capturedusing a digital camera. As shown in FIG. 27, the formulation stainedboth cheeks and the dorsal/ventral surface of the tongue.

Examples 162 and 163

Exposure of Microbes to Barrier-Forming Composition Inhibits CellGrowth: Time-Lapse Microscopy

To determine the inhibitory activity and duration for whichbarrier-forming compositions exhibit activity against microbes,time-lapse analysis was performed on cells exposed to thebarrier-forming composition, compared to untreated bacteria and fungi.

In Example 162, S. mutans microbial cells were exposed to Example 7 forone minute, washed to remove any residual agent, and allowed to grow ina petri-dish containing fresh growth medium. Growth of organisms at 37°C. was monitored for a 6 hour period, and photomicrographs were takenevery 20 minutes over the 6 hour incubation period using a cameraconnected to the microscope.

In control Example 163 the same procedure was followed with untreatedcells.

As shown in FIG. 28, in contrast to the untreated bacteria, where cellsreached confluence by 6 hours, microbes treated with the Example 7barrier-forming composition failed to regrow during the same time periodpost-exposure. Similarly, exposure of Candida cells to the Example 7barrier-forming composition completely inhibited growth during theincubation period (data not shown).

These results further confirmed that the barrier-forming compositionpossesses prolonged antimicrobial activity.

Examples 164-166

In vivo Study: Barrier-Forming Composition (Example 7) Lowers the OralMicrobial Load in Humans: Short- and Long-Term Activity

Short-Term Activity

The duration of activity of Example 7 was determined in healthyindividuals by evaluating the effect of a single application onmicrobial burden of the oral cavity. In Examples 164-166, three healthyindividuals (over 18 years of age, healthy mouth) were enrolled withinformed consent, and asked to apply a single application of thecomposition of Example 7 on their cheeks. A single application wasdefined as three sprays of 0.25 ml each in volume. Next, swabs werecollected from these individuals at baseline (pre-treatment), 1 hour, 2hours, and 6 hours post-treatment. Swabs were cultured on agar mediaplates specific for aerobic or anaerobic organisms, incubated for 24-28hours at 37° C., and the number of CFUs were counted. Effect of Example7 on microbial burden was determined (CFUs), and percentage inhibitionwas calculated for each post-exposure time point relative to thebaseline (0 minutes) CFUs.

The results showed that application of Example 7 led to consistentreduction in microbial load for up to 6 hours (See FIG. 29A, which showsCFUs of a representative tested individual. Treatment with thebarrier-forming composition resulted in 69% to 96% reduction of themicrobial burden in the oral cavity (See FIG. 29B, which shows arepresentative individual's reduction in microbial load.)

Examples 167-169

Long-Term Activity

The activity of the barrier-forming composition over a 5-day periodagainst oral microbes was evaluated. In Examples 167-169, three healthyindividuals were enrolled, and asked to apply a single dosage (threesprays as defined above) of Example 7 three times daily (approximately 9AM, noon, and 3 PM) for a 5-day period (representing a typical 5-daywork-week). Swabs were collected from these individuals at baseline(before application on day 1) and at the end of the day on each dayduring the 5-day period. Collected swabs were cultured on agar mediaplates, incubated for 24-28 hours at 37° C. and at 5% CO₂ humidity, andthe number of CFUs were counted.

The effect of the Example 7 barrier-forming composition on microbialburden was determined (as median CFUs for the three subjects), andpercentage inhibition was calculated for each post-exposure time pointrelative to the baseline (0 min) CFUs. FIG. 30 shows these results in agraph of CFUs versus time (FIG. 30A) and reduction in microbial loadversus time (FIG. 30B). Examples 167-169 demonstrate that application ofExample 7 over 5 days led to consistent reduction in microbial load overthe 5-day test period (FIG. 30A). Treatment with the Example 7barrier-forming composition resulted in 65%-88% reduction of the medianmicrobial burden in the oral cavity of the study participants (FIG.30B).

Examples 170-198

In a clinical study, twenty-nine healthy individuals were enrolled afterinformed consent. Baseline information was recorded (age in years,gender, ethnicity, and date of enrolment). Oral examination of the mouthwas undertaken, and the inside of the mouth (cheek) was swabbed with asterile culture swab. Baseline oral swab samples were cultured todetermine bacterial load prior to study. In Examples 170-198, each ofthe twenty-nine participants were given a spray bottle containing thebarrier-forming composition of Example 7 and instructed to spray theinside of their mouth six times (total volume sprayed is 0.75 ml), thenswish for 30 seconds and swallow. Two groups of participants used theexample barrier-forming composition every two hours, three times a day,for five days (a typical work week). Swabs were collected on days 1, 2,3, and 5 and cultured on media specific for aerobic and anaerobicbacteria. Data were presented as number of microbes: total, aerobic andanaerobic. FIG. 31 shows a graph of total microbial load and breaks downthe total into aerobic and anaerobic counts from just prior to treatmentand on day 5 of treatment. FIG. 32 shows graphs of microbial load overthe 5 day period in oral samples obtained from three representativestudy participants.

Overall, the in vivo testing showed that the barrier-forming compositionexhibits antimicrobial activity against oral microbes, as measured byreduction in the levels of these organisms, over both short- andlong-term duration.

The data showed that treatment with the barrier-forming composition overa 5-day period resulted in reduction in the oral microbial load, fortotal microbes, aerobic and anaerobic organisms.

Example 199-205

Identification of Additional Humectants for Forming a Barrier to PreventMicrobial Penetration

In Example 199 an in vitro filter insert-based model (see FIG. 33) wasused to test different humectants at different concentrations.

Six compositions were prepared according to Table XII based on themixing procedures used for Examples 3-8

TABLE XII Ex. Ex. Ex. Ex. Ex. Ex. Ex. 199 200 201 202 203 204 205Xanthan 0.4 0.4 0.4 0.4 0.4 0.4 Gum Glycerin 4.5 4.5 4.5 4.5 Sorbitol4.5 4.5 4.5 4.5 Xylitol 4.5 4.5 4.5 4.5

Next, 100 μL of Examples 199-205 were placed into filter inserts (poresize 0.8 μm diameter, that allows both bacteria and fungi to passthrough) and allowed to a form a layer. Next, organisms were overlaid onthe layer formed by the test solutions. The filter inserts containingthe layer of test solutions and microorganisms were then placed on thesurface of agar media plates and incubated for 24 hours at 37° C. Afterthe incubation period, the agar media plates were evaluated for growthon filter insert and in the agar media. Growth on filter insert but nogrowth in agar media indicated that the test solution formed a barrier,which prevented the microbes from passing through. In contrast,microbial growth in the filter insert as well as the agar mediaindicated that no such barrier was formed.

The results showed that each of the xanthan gum-based solutionscontaining the tested humectants (singly or in combination) formedintact barriers on the filter insert that prevented the passage ofmicroorganisms into underlying agar medium.

Example 206-213

Determination of the Solubility Limits of Xanthan Gum

To determine the solubility of xanthan gum, it was mixed at differentconcentrations in water and the solubility observed by monitoring thepresence or absence of clumps and free flow of the mixture. Table XIIIreports the results and concentrations.

TABLE XIII Xanthan Gum Example Concentration Solubility 206 0.40%  freeflowing viscous solution 207 0.45%  some clumps, viscous solution 2080.5% more clumps, viscous solution 209 0.6% clumps, more viscous thanabove 210 0.7% clumps, more viscous than above 211 0.8% Extensiveclumps, highly viscous solution, no free flow 212 0.9% Extensive clumps,highly viscous solution, no free flow 213 1.00%  Extensive clumps,highly viscous jelly, no free flow

We found that when mixed at 0.4%, xanthan gum formed a free-flowingsolution (Table XIII). In contrast, mixtures containing 0.45% or 0.5%xanthan gum formed a viscous fluid but contained small clumps. Theextent of clumps increased with increasing concentration of xanthan gum(0.6% and 0.7%). At concentrations ≥0.8%, xanthan gum mixture containedextensive clumps, with a jelly-like consistency and no free flow.

Example 214

Comparison of Cationic CPC in Barrier-Forming Composition with NeutralAntimicrobial Agent in Barrier-Forming Composition

In Example 214, the formulation of Example 7 was made, except theneutral agent Citral was used instead of CPC. The antimicrobial activityof formulations containing CPC (0.1%) or Citral (0.5%) againstStreptococcus was ascertained. The assay described above in Examples48-61 was used to perform these studies.

The results showed that the formulation containing citral exhibitedantimicrobial activity (MIC=12.5%). However, activity of formulationcontaining citral was significantly less potent than that containing CPC(MIC=0.098%).

Example 215

Physico-Chemical Testing of Hydrophobicity and Comparison

In Example 215 thin layer chromatography analysis was used to comparethe hydrophobicity of Example 7 with a hydrophobic composition. Thehydrophobic composition was comprised of the components in Table XIV.

TABLE XIV Wt % Glycerin 7 Sorbitol 5 Poloxamer 338 1 PEG 60 Hydrogenatedcastor oil 1 VP/VA copolymer 0.75 Sodium benzoate 0.5 Cellulose Gum 0.2CPC 0.05 Methyl Paraben 0.05 Propyl paraben 0.05 Sodium Saccharin 0.05Xanthan Gum 0.01 Disodium Phosphate 0.006 Flavoring and coloring agents0.121 *the remainder of the composition was purified water

10 μL of Example 7 and the hydrophobic composition were deposited onpre-made TLC plates (at a distance of 2 cm from the bottom edge). Thespots were air-dried for 5 minutes, and the plates were placed in a TLCchromatography jar containing water as a solvent. The TLC system wasallowed to run until the solvent front reached the top edge of theplate. Plates were removed and the solvent and sample fronts weremarked. The Relative Front (Rf) values were calculated for the twosamples using the formula II:

Rf=Distance travelled by spot/Distance travelled by solvent front  II.

The results showed that the Rf value for the hydrophobic composition andExample 7 were 0.33 and 0, respectively, indicating that the hydrophobiccomposition was highly miscible in water. In contrast, Example 7 did notexhibit any mobility in the aqueous solvent, demonstrating that thisformulation is hydrophobic or not hydrophilic.

It is claimed:
 1. A method for reducing risk of infectious disease in ahost from microorganisms encountered subsequent to application of abarrier-forming composition, the method comprising: administering byspraying or mouthwash delivery, a therapeutically effective amount ofthe barrier-forming composition to a mucosa of the host; the compositioncomprising: about 0.01%≤C≤about 0.4%; 7%≤H≤about 65%; and 0.050%<A;wherein all percentages are by weight of the total composition; whereinC is a carbohydrate gum; H is a humectant; and A is a cationicantimicrobial agents or pharmaceutically acceptable salt thereof;wherein the composition forms a barrier on the mucosa that is active totrap, and kill or neutralize microorganisms; with the proviso that themethod is not administered for prevention or treatment of dentaldisease.
 2. The method of claim 1, wherein the pH of the composition isabout 4 to about
 7. 3. The method of claim 1, wherein the antimicrobial,A, is present in an amount of 0.050%<A<0.1% by weight of the totalcomposition.
 4. The method of claim 1, wherein the composition has abroad spectrum antimicrobial property that is effective to kill orneutralize fungal, bacterial, and viral microorganisms.
 5. The method ofclaim 1, wherein the composition is in the form of a solution.
 6. Themethod of claim 1, wherein the composition is free-flowing and clumpfree.
 7. The method of claim 1, wherein the mucosa is an oral orpharyngeal mucosa.
 8. The method of claim 1, wherein the composition iseffective in 80% of humans to show a decrease of about 50% or greater ofmicrobial load in the oral cavity on the sixth day of three times dailyadministering of the composition.
 9. A method for prevention ofinfectious disease in a host, the method comprising: administering atherapeutically effective amount of a composition to a mucosa of thehost; the composition comprising: about 0.01%≤C≤0.4%; about 7%≤H≤about65%; and 0.050%<A; wherein all percentages are by weight of the totalcomposition; wherein C is a carbohydrate gum; H is a humectant; and A isan antimicrobial agent; wherein the infectious disease is caused by aninfluenza virus.
 10. The method of claim 9, wherein the mucosa is anoral or pharyngeal mucosa.
 11. The method of claim 9, wherein thecomposition is effective in 80% of humans to show a decrease of about50% or greater of microbial load in the oral cavity on the sixth day ofthree times daily administering of the composition.
 12. The method ofclaim 9, wherein the composition is effective to reduce a microbial loadby 65% to 88% in the oral cavity after the administering step.
 13. Themethod of claim 9, wherein the composition has a broad spectrumantimicrobial property that is effective to kill or neutralize fungal,bacterial, and viral microorganisms.
 14. The method of claim 1, whereinthe composition is in the form of a solution.
 15. The method of claim 1,wherein the composition is free-flowing and clump free.
 16. Abarrier-forming composition for a surface treatment comprising: acarbohydrate gum, a humectant, and an antimicrobial agent; wherein thebarrier-forming composition meets the following requirements: about0.01%≤C≤0.4%; about 7%≤H≤about 65%; and 0.050%<A; wherein allpercentages are by weight of the total composition; wherein C is thecarbohydrate gum; H is the humectant; and A is the antimicrobial agent;and wherein the antimicrobial agent is a cationic antimicrobial agent orpharmaceutically acceptable salt thereof; the barrier-formingcomposition is active to trap, and neutralize or kill microorganisms;wherein the barrier-forming composition has a viscosity of less than 500cps; wherein the pH of the barrier-forming composition is about 4 toabout
 7. 17. The barrier-forming composition of claim 16, wherein thecomposition is in the form of a solution.
 18. The barrier-formingcomposition of claim 16, wherein the composition is free-flowing andclump free.
 19. The barrier-forming composition of claim 16, wherein thecomposition has a broad spectrum antimicrobial property that iseffective to kill or neutralize fungal, bacterial, and viralmicroorganisms.
 20. The barrier-forming composition of claim 16, whereinthe antimicrobial, A, is present in an amount of 0.050%<A<1% by weightof the total composition.